Project Summary/Abstract The Golgi complex is the central sorting station for nearly a third of all proteins in eukaryotic cells, but how cells regulate the flow of material through this organelle remains unknown. Secretory traffic must pass through the Golgi to be fully glycosylated and proteolytically processed. The Golgi must successfully traffic hundreds of membrane and lumenal proteins to several different sub-cellular destinations including the plasma membrane, endosomes, lysosomes, and the endoplasmic reticulum. Protein and membrane traffic into and out of the Golgi is controlled by GTPases of the Arf and Rab families that function by recruiting effector proteins to generate, transport, and tether membrane vesicles and tubules. The master regulators of these essential GTPase pathways are the GEF (guanine-nucleotide exchange factor) proteins that must ?decide? where and when to activate their substrate GTPases. For most of these GEFs, we do not know how the localization, timing, and magnitude of their activity is regulated. Therefore, we do not fully understand the molecular logic of Golgi trafficking. Our lab has focused on deciphering what cellular signals the Golgi GEFs are listening to, and how the GEFs interpret these signals. We have discovered that several of these GTPase trafficking pathways communicate with each other through protein-protein interactions in which an activated GTPase positively regulates the GEF of another GTPase. One important implication of this finding is that activation of the distinct pathways are coordinated. Although we have uncovered regulatory mechanisms for some of the Golgi GEFs, the others remain poorly understood, and the overall molecular logic of these pathways is only beginning to come into focus. The primary question our proposed research program seeks to answer is: How do the Golgi GEFs make molecular decisions? We will address this question by investigating each of the Golgi GEFs with a broad set of tools, utilizing biochemical reconstitution reactions, structural biology, in vivo functional assays, live-cell imaging, and genetic experiments. We will use biochemical approaches to determine the precise roles of GEF regulatory subunits and domains. We will use structural approaches to visualize each GEF ?caught in the act? of performing nucleotide exchange on its GTPase. Mutants generated based on hypotheses arising from biochemical and structural experiments will then be investigated in vivo to determine the consequences of perturbing specific interactions and domains. We will use unbiased proteomic and genetic approaches to reveal the identity of unknown GEFs and to discover unknown regulators of the Golgi GEFs. We will use established cell biological approaches to determine the physiological importance of the regulatory mechanisms. Taken together, the combined results of in vivo and in vitro experiments will enable us to determine the mechanisms the GEFs use to localize to their site of action, identify their substrate, and regulate their activity. Therefore, we will define how the Golgi GEFs sense and integrate signals, we will obtain a holistic view of how they work together, and we will uncover the molecular logic underpinning how the Golgi functions.